Neurofibromatosis Type 1 (NF1; OMIM #162200) is the most common of autosomal dominant genetic disorders, affecting 1 in 3500 individuals worldwide (Riccardi et al. Am J Pathol 1994; 145:994-1000; Gutmann et al. JAMA 1997; 278:51-7). Currently more than 100,000 Americans suffer from NF1. NF1 is a tumor predisposition syndrome. The most common tumor pathologies in afflicted individuals are neurofibromas (100% incidence), malignant peripheral nerve sheath tumors (MPNST, 15% incidence), and optic gliomas (20% incidence). The NF1 patients may also have other issues, including cognitive deficits in children. In addition to neurofibromas which may occur in the nervous tissue, skin, bones, and muscles, the disease is also associated with hyperpigmented spots.
Multiple neurofibromas are the hallmark of NF1. One subtype of neurofibroma, the plexiform neurofibroma, occurs in about 30% of NF1 individuals. NF1 individuals with plexiform neurofibromas have up to 10% lifetime risk of developing malignant peripheral nerve sheath tumors (Evans et al. J Med Genet 2002; 39:311-4), the most common malignant tumors associated with NF1. Surgery is the only treatment for plexiform neurofibromas and MPNST; however complete surgical resection is often not possible. The prognosis for patients with MPNST is poor, with an overall 5-year survival rate of just 34% (Ducatman et al. Cancer 1986; 57:2006-21). Despite the urgent need, targeted therapies for MPNST to improve survival are not obvious.
Loss-of-function mutations in the NF1 tumor suppressor gene (often referred to herein as neurofibromin, the NF1 gene, or simply NF1) underlie these disease phenotypes. The NF1 tumor suppressor gene is often referred to herein interchangeably as the neurofibromin gene, the NF1 gene, or simply NF1, as will be easily understood by the skilled artisan. The skilled artisan will also understand that NF1 may refer to the disease state, i.e., the state of having Neurofibromatosis Type 1. The NF1-encoded protein, neurofibromin, has been shown to function as a Ras-GTPase activating protein (GAP). Neurofibromin converts Ras from its active GTP to its inactive GDP isoforms. Loss of function at NF1 dysregulates Ras oncogenic signaling pathway thus promotes abnormal cellular proliferation and tumorigenesis (Bollag G., and McCormick F. Nature 1992; 356:663-4). Many NF1-deficient tumors contain elevated levels of RAS-GTP and/or show upregulation of the RAS-dependent signaling pathway, supporting the notion that deregulated RAS signaling may contribute to tumor development (DeClue et al. Cell 1992; 69:265-73); Basu et al. Nature 1992. 356:713-5; Bollag G., et al. Nat Genet 1996. 12:144-8; Feldkamp et al. Surg Neurol 1999. 51:211-8; Ingram D A., et al. J Exp Med 2001.194:57-69). It is generally accepted that Schwann cells are the primary transformed cells in neurofibromas, and that loss of heterozygosity (LOH) of the NF1 locus in Schwann cells initiates tumorigenesis and development of neurofibromas. LOH of the NF1 locus has been found only in Schwann cells, but not in other cells in NF1 neurofibromas and MPNST (Kluwe L., et al. Genes Chromosomes Cancer 1999. 24:283-5; Rutkowski J L., et al. Hum Mol Genet 2000. 9:1059-66; Legius E., et al. Nat Genet 1993. 3:122-6). Mice with Schwann cell lineage specific ablation of the Nf1 gene develop neurofibromas (Zhu Y, et al. Science 2002. 296:920-2; Zhang L., et al. Proc Natl Acad Sci USA 2006. 103:9136-41; Wu J., et al. Cancer Cell 2008. 13:105-16). While the cells involved in NF1 neurofibroma tumorigenesis have been identified, little is known about the contributing gene networks, signaling pathways, and their upstream regulatory networks.
The fact that loss-of-function mutations in NF1 occur in both neurofibromas and MPNST suggests that NF1 mutations alone are not sufficient for neurofibromas to progress to MPNST. Recent research shows that certain neurofibroma subtypes accumulate additional changes, such as those affecting the p19ARF-MDM2-TP53 and p16INK4A-Rb signaling cascades, resulting in their transformation into MPNST (Carroll, and Ratner. Glia 2008.56:1590-605). Deletions or other types of mutations in the p53 locus have been found in 29% to 75% of NF1 MPNST (Menon, et al. Proc Natl Acad Sci USA 1990. 87:5435-9); Legius, et al. Genes Chromosomes Cancer 1994. 10:250-5; Rasmussen, et al. Genes Chromosomes Cancer 2000. 28:425-31). Consistent with a role of p53 in the progression of MPNST, mice that harbor both Nf1 and Tp53 mutations develop MPNST (Cichowski, et al. Science 1999. 286:2172-6).
A few other gene changes, e.g. EGFR, TWIST1 and SOX10, have been implicated (Tabone-Eglinger, et al. Sarcoma 2008; 2008:849156; Miller, et al. Cancer Res 2006. 66:2584-91). However, there is less information on the regulatory networks related to NF1 MPNST transformation.
MicroRNAs (miRNAs) are potential upstream regulators of NF1 tumorigenesis and progression. miRNAs are a class of small non-coding RNAs of about 19-25 nucleotides that function as negative post-transcriptional gene regulators; and can regulate the entire set of genes (Lim, et al. Nature 2005. 433:769-73). miRNAs hybridize to the 3′ untranslated region (UTR) of target mRNAs and repress translation or mediate mRNA cleavage. miRNAs provide important regulatory functions in a variety of biological processes, including development, cell proliferation, differentiation, and apoptosis. Studies show that miRNAs critically regulate tumorigenesis and progression by targeting oncogenes, tumor suppressor genes, or genes related to proliferation, angiogenesis, and apoptosis (Hwang, et al. Br J Cancer 2006. 94:776-80; Hwang, et al. Br J Cancer 2007. 96 Suppl:R40-4). Different tumor types and tumors at various stages of differentiation exhibit unique miRNA profiles (Rouhi, et al. Mamm Genome 2008. 19:517-25; Visone, et al. Am J Pathol 2009. 174:1131-8).
Presently, there are no curative treatments for NF1 and NF1-tumors. Accordingly improved methods of diagnosing those at risk for developing NF1 and for treatment of NF1 would be of great benefit.